We disclose herein DNA vectors useful for genetic engineering manipulations of a wide range of gram-negative bacteria, and methods for genetically modifying a wide range of gram-negative bacteria using the vectors disclosed herein.
The field of genetic engineering of microorganisms, including recombinant DNA technology, has developed in large measure on the basis of a vast store of detailed, basic knowledge of the genetics of Escherichia coli (hereinafter "E. coli") and an extensive array of DNA vectors, plasmids, and phages specifically developed with the aid of such knowledge for use in E. coli. Although E. coli is itself classified as a gram-negative bacterium, most of the DNA vectors and phages developed for use in E. coli are unsuitable for use in other gram-negative bacteria, outside a relatively limited group of bacteria closely related to E. coli, for example, Salmonella. Nevertheless, there are many gram-negative strains of current commercial utility, the genetic modification of which will provide substantial economic benefit. Examples of such gram-negative strains include members of the following genera: Rhizobium, Agrobacterium, Pseudomonas, Klebsiella and Azotobacter.
There are several barriers that prevent the direct use of E. coli vectors and phages in a broad range of gram-negative bacterial strains. One such barrier is the fact that most of the plasmids commonly used as vectors in E. coli are unable to replicate normally in most other gram-negative strains. Another barrier is that efficient plasmid transfer by conjugation between E. coli and other gram-negative strains, or between other gram-negative strains, does not occur with most widely used E. coli vectors. Furthermore, other means for introducing DNA into most gram-negative strains are poorly developed outside of E. coli and its close relatives (hereinafter referred to as the E. coli group). Transformation of gram-negative strains outside of the E. coli group has been demonstrated in some instances; however, the reported efficiencies of transformation have been much lower in most cases than those obtainable with E. coli. While it is possible that higher frequencies of transformation could be obtained for individual gram negative strains by extensive trial and error modification of transformation conditions, it would be desirable to develop vectors that can be transferred by general means applicable to a wide range of gram-negative bacteria.
The drug-resistance plasmid RP4 is known to be transferable across a broad range of gram-negative host bacteria [Datta, N., et al., J. Bact. 108, 1244 (1971); Datta, N., and R. W. Hedges, J. Gen. Microbiol. 70, 453(1972); Olsen, R. H., and P. Shipley, J. Bact. 113, 772 (1973); and Beringer, J. G., J. Gen. Microbiol. 84, 188 (1974)]. Modifications to RP4, to make it useful for genetic manipulation of gram-negative strains, have been reported in the prior art. In particular, RP4 has been modified for use as a vehicle for transposon mutagenesis. Beringer, J. E. et al., Nature 276, 633 (1978) reported the transfer of the transposon Tn5 to Rhizobium using pPH1, a broad host range plasmid of the IncP incompatibility group containing Tn5 and DNA of bacteriophage Mu inserted into the plasmid. Transfer was effected by conjugation between E. coli carrying pPH1 (with inserted Mu and Tn5) and a recipient Rhizobium strain. The effect of the Mu insertion was to render the plasmid unstable in the recipient strain so that the transferred plasmid was ultimately eliminated. The transposon could be rescued in recipient cells in which a translocation had occurred prior to elimination of the plasmid. Plasmids constructed in this manner have been termed "suicide plasmids." The use of such plasmids to introduce random nutations into Rhizobium strains has been reported by Meade, H. M., et al., J. Bact. 149, 144 (1982), and in Agrobacterium by VanVliet, F. B., et al., Plasmid 1, 446 (1978).
A number of difficulties have been found to be associated with the use of RP4, or other plasmids of incompatibility group P, carrying the Mu genome and Tn5 in transposon mutagenesis. In some instances, the yield of transconjugants was very low, as measured by the acquisition of a drug resistance associated with the transposon. In many cases, the frequency of a transferred drug resistance was not significantly higher than the spontaneous resistance frequency. In other cases, stably replicating derivatives of the "suicide plasmid" arose, presumably by a deletion of the Mu insert. Such mutant plasmids simulate Tn5 transposition events and it is very time consuming to distinguish between Tn5 insertions and other phenomena, such as the acquisition of a stably replicated plasmid. Furthermore, it was frequently the case that mutations occurred, not only by transposon insertion into the recipient genome, but also by transfer of the Mu phage DNA from the plasmid to the recipient genome. Mutations caused by Mu insertion occurred at sites remote from the transposon insertion site and could not be cloned subsequently, since no readily identifiable marker was associated with a Mu insertion.
In order to facilitate discussion of the invention, the following definitions are provided:
Replicon: a fundamental unit of replication comprising all the genetic elements sufficient to confer autonomous replication in a bacterial cell, together with the DNA whose replication is controlled thereby. The bacterial chromosome, plasmids and phage DNA's are examples of replicons existing in a bacterial cell. Individual replicons differ in the extent to which they are functional in different host cell species. Many of the replicons commonly employed as plasmids for genetic engineering are functional only in bacteria of the E. coli group. Others, such as RP4, are able to replicate in a wide range of gram-negative bacterial hosts.
oriT: Site of origin of transfer replication. The mechanism of DNA transfer by bacterial conjugation includes a replication of the plasmid in which a break is introduced into one strand of duplex plasmid DNA, DNA replication then commences at the site of the break, together with transfer of the cut strand to the recipient cell. Some authors have used the designation nic, to indicate the site of the single-stranded break which initiates transfer replication. The term nic is considered to be equivalent to oriT.
Mob-site: A genetic locus necessary for mobilization of a plasmid transferrable by bacterial conjugation. The Mob-site is believed to include oriT. The Mob-site is considered to be the target locus of certain trans-acting functions coded by tra genes. The existence of a Mob-site is a necessary condition for transfer; however, the tra functions must also be provided. Since the latter act in trans, the genes which code for them may be located elsewhere in the cell; for example, on another replicon. Tra functions and Mob-sites also differ with respect to host range. For example, the tra functions and Mob-site of the F factors are limited in function to conjugal transfers between members of the E. coli group. By contrast, the tra functions and Mob-site of the plasmid RP4 permit its conjugal transfer over an exceedingly wide range of gram-negative organisms.
Mob-site segment: As defined herein, the term "Mob-site segment" means that portion of a plasmid which includes the Mob-site but lacks DNA encoding operative tra functions or replication functions.
The genetic analysis and isolation of genes have been greatly facilitated in recent years by the use of transposons. Transposons are special DNA segments which have certain structural features and carry within them certain genes which enable them to be transferred as a unit in a random fashion from one genetic locus to another, with a characteristic frequency. Typically, a transposon will contain one or more drug-resistance genes. These provide convenient selection markers to identify the presence of the transposon and to facilitate cloning of any DNA segment containing a transposon. Insertion of a transposon may occur within a gene, resulting in loss of function for that gene. Transposon mutagenesis, combined with restriction site mapping and cloning provides an extremely powerful and rapid technique for genetic and physical analysis of an organism, together with the ability to clone a desired gene of the organism. Until recently, the techniques of transposon mutagenesis and molecular gene cloning have been restricted to E. coli and closely related organisms.
The techniques have been employed, as described, supra, in a variety of other gram-negative bacteria. The prior art attempts to apply these techniques generally have been limited by the difficulties previously described. The present invention discloses the construction and use of new vector plasmids that facilitate in vivo manipulation by transposon mutagenesis and provide techniques for site-specific introduction of a foreign gene or transposon. These techniques are generally applicable across a wide range of gram-negative organisms.